The action of a negative allosteric modulator at the dopamine D(2) receptor is dependent upon sodium ions

Sodium ions (Na(+)) allosterically modulate the binding of orthosteric agonists and antagonists to many class A G protein-coupled receptors, including the dopamine D(2) receptor (D(2)R). Experimental and computational evidences have revealed that this effect is mediated by the binding of Na(+) to a conserved site located beneath the orthosteric binding site (OBS). SB269652 acts as a negative allosteric modulator (NAM) of the D(2)R that adopts an extended bitopic pose, in which the tetrahydroisoquinoline moiety interacts with the OBS and the indole-2-carboxamide moiety occupies a secondary binding pocket (SBP). In this study, we find that the presence of a Na(+) within the conserved Na(+)-binding pocket is required for the action of SB269652. Using fragments of SB269652 and novel full-length analogues, we show that Na(+) is required for the high affinity binding of the tetrahydroisoquinoline moiety within the OBS, and that the interaction of the indole-2-carboxamide moiety with the SBP determines the degree of Na(+)-sensitivity. Thus, we extend our understanding of the mode of action of this novel class of NAM by showing it acts synergistically with Na(+) to modulate the binding of orthosteric ligands at the D(2)R, providing opportunities for fine-tuning of modulatory effects in future allosteric drug design efforts.